The present invention is directed to a potent tight-binding multisubstrate adduct inhibitor of glycinamide ribonucleotide transformylase (GAR TFase; E.C. 2.1.2.2). GAR TFase is a crucial, reduced folate-requiring enzyme involved early in de novo purine biosynthesis, catalyzing the formyl transfer from (6R, alpha-S)-10-formyl H.sub.4 folate to glycinamide ribonucleotide (GAR). It has thus attracted some interest as a target enzyme for the design of pharmacologically active substances, especially anti-neoplastic agents. See, Chabner, B.A., et al., in "Chemistry and Biology of Pteridines, Proc. 8th International Symposium," Cooper and Whitehead, eds. pp. 945-51, deGruyter, Berlin, 1986.
The inhibitor compounds disclosed herein will be useful as an anti-gout and/or anti-neoplastic therapeutic agent or as a potentiator for other such agents.
Specific potent inhibitors of enzymes have previously been designed using the ideas of Pauling and Jencks, which stress the importance of the enzyme's ability to stabilize a substrate's passage through its transition state to product. Much of this stabilizing energy is derived from the binding energy acquired when substrate combines with enzyme. See for example, L. Pauling, Chem. Eng. News, 24, 1375 (1946); W. P. Jencks, Chemistry and Enzymology, (Dover Publications, Inc., New York, 1987) and Gandour et al., "Transition States of Biochemical Process", (Plenum Press, New York, (1978); Collins et al., J. Biol. Chem., 246, 6599-6605 (1971); Bartlett et al., J. Am. Chem. Soc., 106, 4282-4283 (1984); Chan et al., Heterocycles, 23, 3079-3085 (1986) and Park et al., J. Med. Chem., 22, 1134-1137 (1979); Wolfenden, Annu. Rev. Biophys. Bioeng., 5, 271-305 (1976) and Wolfenden, Acc. Chem. Res., 5, 10-18 (1972).
A number of potent specific inhibitors of enzymes have been designed using the concept of multisubstrate adduct inhibition (or MAI). See, Gandour and Schowen, eds., "Transition States of Biochemical Processes," Plenum Press, New York, 1978; and Broom, Federation Proc., 45, 2779-2783 (1986). For a recent list of specific enzyme inhibitors, see Wolfenden, et al., in "Enzyme Mechanisms", Page and Williams, eds., pp. 97-102, Royal Society of Chemistry, London, 1987.
The tying together of both substrates of a bimolecular, enzyme-catalyzed reaction yields a molecule possessing the binding stabilization of both individual substrates, in addition to the entropic advantage of reduced molecularity (Jencks, W.P., Advances in Enzymology, 43, 219-410 (1975)). However, it should be noted that a multisubstrate adduct inhibitor is not intended to mimic the transition state of a catalyzed reaction.
The degree to which an enzyme-inhibitor complex remains associated with the desired substrate is a measure of the inhibitor's potency. A common measure for the effectiveness of an inhibitor is its dissociation constant, K.sub.D, or its inhibition constant, K.sub.I. To the first approximation, these are the same, and are a ratio of free inhibitor and enzyme to the enzyme inhibitor complex. The smaller the number, the less free enzyme is present, and the better the inhibitor.
Prior to the present invention, other inhibitors of GAR TFase have been produced, but none were as potent in vitro as the commonly used anti-folate agent methotrexate (which is specific for another enzyme important to purine biosynthesis, dihydrofolate reductase [DHFR]). The highly active GAR TFase inhibitor of the present invention thus represents a breakthrough in this area.
There are a modest number of compounds actually tested against GAR TFase. Those with published K.sub.I values (inhibition constants) include the work of Caperelli et al., (J. Med. Chem., 29, 2117-2119 (1986) and J. Med. Chem., 30, 1254-1256 (1987)) who have shown that the substitution at N.sup.10 of DDF with various substituted alkyl, acyl, benzyl, and heterocyclic groups produce modest inhibition of murine lymphoma GAR TFase, with K.sub.I 's ranging from 1.3 to 33 uM. These compounds were also shown to inhibit thymidylate synthase (TS) and dihydrofolate reductase (DHFR), illustrating a great lack of specificity for GAR TFase. See, Jones et al., J. Med. Chem., 28, 1468-1476 (1985).
The inhibitor of the present invention is conservatively about 10.sup.5 times more potent (based on a comparison of the K.sub.I values) than the above-mentioned compounds and has no significant inhibitory effect on DHFR or TS. ##STR2## appears to be an inhibitor of GAR TFase. This inhibitor, when tested against solid tumors in mice, was indirectly shown to inhibit GAR TFase and to cause depletion of intracellular pools of ATP and GTP, end products of purine biosynthesis. See, Taylor et al., J. Med. Chem., 28, 914-921 (1985); Moran et al., Proc. Amer. Assoc. Cancer Research, 26, 231 (1985) and Beardsley et al., "Chemistry and Biology of Pteridines, Proc. 8th International Symposium," B. A. Cooper and Whitehead, V. M., eds. (deGruyter: Berlin, pp. 953-7 (1986).
Since no data was reported for the activity of DDATHF against purified GAR TFase (i.e., there is no K.sub.I given) it is difficult to compare DDATHF to the inhibitors of the present invention in terms of potency. DDATHF appears to show impressive activity against a variety of solid tumors in mice whereas methotrexate (MTX), a common anti-folate in use today, shows minimal activity against these same tumors. Beardsley et al., Chemistry and Biology of Pteridines, 53-957 (1986).
None of the previously discussed inhibitors are multisubstrate adduct inhibitors. The series of DDF analogues tested by Caperelli et al., were poor inhibitors of GAR TFase both in respect to their potency and specificity. The compound of Taylor et al. may have sites of action other then GAR TFase and a quantitative account of its activity against a purified GAR TFase has yet to be reported.
Two previous attempts at the synthesis of a multisubstrate adduct inhibitor for GAR TFase have been reported. For example, Licato, Jr., in his Ph.D. Dissertation (U. Utah) reported the unsuccessful efforts toward the synthesis of the following compound: ##STR3## See, Diss. Abstr. Inc., B, 47, 2918 (1987).
In J. Med. Chem., 31, 697-700 (1988), Temple and his coworkers reported the synthesis of several potential anticancer agents which were designed to be effective as GAR TFase inhibitors. These tetrahydrofolic acid (THF) derivatives included the following: ##STR4## No useful biological activity was reported for any of these compounds against GAR TFase.
The synthetic approach of the present invention, has been found capable of generating the most potent and specific inhibitor of the GAR TFase enzyme yet described anywhere in the literature.